The present invention relates to methods of immunoassay of antigens and to kits for carrying out such methods. In particular, it relates to methods of immunoassay employing enzyme labels (hereinafter referred to as enzyme immunoassays) whereby two antigens are assayed simultaneously in a single sample.
The term "antigen" as used herein will be understood to include both permanently antigenic species (for example proteins, peptide hormones, bacteria, bacteria fragments, cells, cell fragments and viruses) and haptens which may be rendered antigenic under suitable conditions (including, for example, narcotics, hypnotics, analgesics, cardiovascular drugs, vitamins, non-peptide hormones and metabolites thereof, antibiotics, pesticides and sugars).
Radioimmunoassays which can simultaneously measure two different analytes in a single sample have been available for several years. The principle of these dual analyte or "combo" assays is that two conventional assays are performed simultaneously in the same reaction vessel, with the two component assays using different radionuclide labels which can be distinguished from one another by their different energy levels. Suitable pairs of radionuclides which have been employed include .sup.125 I/.sup.131 I and .sup.125 I/ .sup.57 Co. Such dual radioimmunoassays offer significant benefits in terms of convenience, speed of obtaining results, laboratory throughput etc. and have become widely accepted in those areas where it is routine practice to measure two analytes in a single sample (e.g. vitamin B.sub.12 and folate for the differential diagnosis of certain anaemias). However, these assays suffer from the same inherent disadvantages as all assays using short-lived radionuclides as labels. These include short shelf-life, exposure of the user to radiation, the requirement for special facilities and problems with disposal of waste.
The use of non-radioactive labels overcomes these problems and currently enzyme labels are the most commonly used replacement for immunoassays. For enzymes to be used in "combo" assays, it is necessary to identify two suitable enzyme-substrate pairs which not only fulfil the criteria necessary for enzyme immunoassays (ability to be conjugated to one of the reactants with little or no loss of enzyme or immunological activity, freedom from interference by the sample or assay conditions etc) but which, under certain conditions, do not interact with one another during the immunoreaction and can simultaneously catalyse separate substrate conversions to generate products which can be measured independently of one another, either by direct monitoring of their production or by monitoring removal of substrate during the incubation period.
Blake et. al. in Clinical Chemistry (1982) 28 1469-1473 reported the development of a dual analyte enzyme immunoassay for the two haptenic hormones thyroxine (T.sub.4) and triiodothyronine (T.sub.3), involving the use of alkaline phosphatase and .beta.-galactosidase as the enzyme labels and phenolphthalein monophosphate and o-nitrophenyl-.beta.-galactoside (o-NPBG) as the respective substrates. In this assay, firstly unlabelled T.sub.3 and T.sub.4 compete respectively with T.sub.3 -.beta.-galactosidase conjugate and T.sub.4 -alkaline phosphatase conjugate for binding to an antibody reagent comprising T.sub.3 and T.sub.4 specific antibodies. The bound fractions of the two enzyme labels are separated by a second antibody precipitation and, after washing, the precipitate is resuspended in an enzyme substrate solution containing phenolphthalein monophosphate and o-nitrophenyl-.beta.-galactoside. The amounts of the two enzyme labels are then determined sequentially in a two-stage incubation protocol, the amount of .beta.-galactosidase being initially determined by monitoring the absorbance of o-nitrophenol at 420 nm and the pH then being raised to determine the amount of alkaline phosphatase by monitoring the absorbance at 540 nm. Since the two enzyme reactions are performed sequentially rather than simultaneously, this assay is not a true "combo" immunoassay. We have now found, however, that it is in fact feasible to perform true "combo" immunoassays employing two enzyme labels.